|The autism spectrum disorder Rett syndrome (RTT; Online
Mendelian Inheritance in Man #312750) is the leading cause of
intellectual disabilities in girls with an incidence of 1:10,000 worldwide
[1,2]. RTT cases occur sporadically with an extremely low familial rate
[3,4]. Individuals with RTT develop typically until 6-18 months when a
constellation of neurological hallmarks appears. Besides autistic features
such as aberrant visual and aural contacts, characteristic symptoms that
distinguish from classical autism include stereotypic hand movements,
motor coordination deficits, breathing abnormalities, seizures and loss
of acquired speech as well as purposeful hand use [5,6]. Underlying
these traits are mutations of the gene MECP2 located in chromosome
Xq28 that encodes MeCP2 (methyl-CpG-binding protein 2) in its two
isoforms (e1 and e2) following alternative splicing of its four exons [7-
9]. In this review, we will briefly discuss current perspectives on MeCP2 function, and then will describe in detail several mouse models of
RTT based on loss-of-function of Mecp2 and their use for establishing
rescue models (Figure 1, Table 1), wherein we pay close attention to
behavioural and morphological phenotypes. As to electrophysiological
manifestations, we refer the readers to a recent review .
|MeCP2 is a transcription factor that binds methylated Cytosine
in C-p-G islands in DNA, controlling gene expression and chromatin
remodeling [11-13]. MeCP2 dysfunction as a result of pathogenic
mutations (i.e. missense, nonsense, truncations due to premature STOP
codons) accounts for most RTT cases. As an X chromosome-linked
disorder, complete absence of functional MeCP2 causes early mortality
in boys, who hardly survive over 2 years. Females live much longer
owing to the presence of a considerable amount of functional MeCP2
produced by the allele in the non-inactivated X chromosome ,
albeit reduced by approximately 50% from typical levels (i.e. mosaicism
of X chromosome inactivation).
|Normal MeCP2 function requires two major domains, the methyl-
CpG binding domain (MBD) and the transcriptional repression
domain (TRD). MeCP2 was initially thought to primarily play an
inhibitory role in gene expression by recruiting chromatin remodeling
protein complexes and transcriptional co-repressors like mSin3a
and Histone Deacetylases (HDACs) . This initial view of MeCP2
function as a transcriptional repressor has been extended recently to
include an activating function, as one of the significant findings from a microarray study demonstrated that the majority of affected genes are
activated in MECP2 overexpressing mice and down-regulated in Mecp2 null mice . However, only a small number of genes were selected
in that study to confirm MeCP2 binding specificity, and the possibility
that MeCP2 might indirectly promote gene expression via regulation of
gene silencers such as REST/CoREST  or microRNAs [18,19] has
not been formally excluded. Furthermore, the changes observed in the
Chahrour et al. , microarray study and others [20,21] are usually
small in magnitude, which suggests that even though directly bound to
targeted gene, MeCP2 might not act as a classical transcriptional factor
but rather as a fine-tuning gene regulator.
|Research on RTT has been mostly focused on neuronal
abnormalities, such as reduced cell body size, delayed dendritic
maturation and synaptogenesis, reduced dendritic branching and
axonal arborization, altered spine and presynaptic bouton density
(see detailed discussion below for each mouse model). These findings led to the assumption that RTT originates exclusively from neuronal
dysfunction. However, this traditional view has been questioned by
recent observations of impaired morphology in wildtype neurons
when co-cultured with MeCP2-deficient glial cells . Consistently,
restoration of MeCP2 levels in glial cells in vivo significantly alleviated
several RTT-like behaviors in Mecp2 null mice, as well as improving
neuronal morphology . Such non cell-autonomous effects on
neurons are also displayed by MeCP2-deficient microglia, which release
high levels of glutamate that are deleterious to co-cultured neurons .
Consistently, transplants of wildtype bone marrow to Mecp2 null mice
arrested the progression of RTT-like features due to the generation of
myeloid cells that differentiated into a microglial phenotype .
|Another classical notion is that MeCP2 is indispensable for
normal brain development, which infers that irreversible neurological
dysfunction would occur in the absence of MeCP2 from an early age.
However, recent studies found that re-activating the endogenous Mecp2 gene at normal levels in mature fully symptomatic Mecp2 null mice
reverts several RTT-like phenotypes . Such observations indicate
that MeCP2’s function goes beyond early brain development and may
include additional roles in neuronal maintenance throughout the whole
life . More provocative findings on MeCP2 function continue to
come out; however, none of those discoveries would take place without
the use of current experimental mouse models, as discussed below, and
those to come in the future.
|Widespread Mecp2 Deletions
|Mecp2tm1.1B mice (a.k.a. Bird nulls)
|The Mecp2tm1.1B mouse line was generated in the laboratory of Adrian Bird by crossing females with Mecp2 exons 3 and 4 flanked
by loxP sites (Mecp22loxB) with males ubiquitously expressing the Cre transgene to delete the loxP-flanked exons . It should be noted that
Mecp22loxB mice already have ~50% lower levels of MeCP2 than wildtype
controls (before Cre-mediated recombination), which cause significant
neurological symptoms similar to Mecp2 null mice, albeit less severe;
thus they are considered a hypomorph Mecp2 allele [29,30]. The
progeny after Cre-mediated recombination and deletion of loxP flanked
exons 3 and 4, however, completely lacks Mecp2 mRNA and protein
expression [28,31]. Hemizygous null mice (i.e. Mecp2-/y males without
an X chromosome) do not show any overt phenotypes until 4-6 weeks
of age, but rapidly develop several neurological deficits and typically die
at 7-8 weeks. Neurological deficits are usually homogenous, including
inertia, ataxia, hindlimb clasping when suspended by the tail, irregular
breathing, and reduced body weight. It should be noted that these
features were observed in mice with a C57BL/6 genetic background.
When these mice were crossed to the 129 strain, pronounced weight
changes were not observed, but the remaining phenotypes remained
unaffected. Heterozygous female mice (i.e. with a mosaic expression of
wildtype and null alleles) develop similar RTT-like symptoms, but they
have a conspicuously slower and milder disease progression.
|Brain specific Mecp2 deletion was obtained by breeding Mecp22loxB
mice with Nestin-Cre transgenic mice, which deleted Mecp2 exons
3 and 4 in the neuronal and glial cell lineage. These mice displayed
a phenotype similar to the widespread MeCP2 deficiency, which
supports the notion that RTT originates from its impaired function
in the brain. Neuropathological examination of Mecp2tm1.1B mice in the symptomatic stage did not reveal any major abnormalities such as
disorganized cortical lamination and ectopias. However, more detailed
analyses showed that cortical growth is stalled as symptoms appear,
and that neurons are smaller and more packed at a higher density in
layer II/III of somatosensory and motor cortices . In these cortical
layers, the length of apical dendrites and dendritic spine density are
lower compared to wildtype mice. These impairments are accompanied
by the occurrence of immature dendrites and dendritic spines also
in layers V and VI of the motor cortex . In motor cortex layer V,
however, diverse synaptic phenotypes were found: spine numbers are
largely decreased in apical dendrites but not basal dendrites [34,35].
The immunoreactivity levels of the presynaptic marker VGLUT1
(vesicular glutamate transporter-1) and the postsynaptic marker PSD95
(postsynaptic density-95) are lower in hippocampal neurons grown in
autaptic microisland cultures . Intriguingly, the density of VGLUT1
puncta is lower in hippocampal area CA1 of perfusion-fixed Mecp2tm1.1B
mice than in wildtypes, but only at 2 weeks of age (presymptomatic
stage), and not 5 weeks when overt symptoms start to appear , which
again suggest that Mecp2tm1.1B mice have diverse synaptic phenotypes in
different brain regions.
|In the olfactory system, primary sensory neurons of Mecp2tm1.1B
mice have impairments in axonal fasciculation, path-finding, and
terminal differentiation [37,38], but on the postsynaptic side, no
abnormalities in the mitral cells were observed . The consequences
of MeCP2 deficiency in Mecp2tm1.1B mice are also manifested in
postnatal neurogenesis processes, including delayed maturation of
newly-born neurons, altered expression of presynaptic proteins, and
reduced formation of dendritic spines . However, it does not affect
the generation of immature neurons, which is in agreement with an
early conclusion that MeCP2 is not involved in neuronal differentiation
but only in neuronal maturation and maintenance in a cell-autonomous
|Mecp2tm1.1J mice (a.k.a. Jaenisch mutants)
|Unlike the complete loss of MeCP2 protein expression in
Mecp2tm1.1B mice, the deletion of only exon 3 in Mecp2tm1.1J mice leads
to the expression a protein fragment lacking part of the MBD but
with an intact but seemingly non-functional C-terminus . Indeed,
these mice (in either 129, C57BL/6, or BALB/c backgrounds) show a
very similar neurological phenotype to Mecp2tm1.1B mice, although
slightly milder and with a longer lifespan (10-12 weeks). Brain-specific
Mecp2 exon 3 deletion by recombination of floxed Mecp2 (Mecp22loxB)
with Nestin-Cre also results in phenotypical features identical to
that of widespread whole-body deletion, as in Mecp2tm1.1J mice. At
the neuropathological level, Mecp2tm1.1J mice also have reduced brain
weight and neuronal size .
|More detailed analyses of different brain regions at the cellular
level revealed that both mice have similar characteristics, but that
Mecp2tm1.1B mice generally exhibit a more severe phenotype [44,45].
For example, dendritic spine density of all principal neurons of the
hippocampus is lower in Mecp2tm1.1B mice than in wildtype controls,
while this feature is only apparent in area CA1 of Mecp2tm1.1J mice .
However, a recent study showed that CA1 pyramidal neurons in the
hippocampus of Mecp2tm1.1J mice have lower dendritic spine density and
smaller asymmetric spine synapses than wildtype controls, but only in
the presymptomatic stage .
|Mecp2tm1Tam mice lack the entire MBD sequence, and carry a nonfunctional splicing site that prevents splicing and transcription of
the downstream Mecp2 sequence, leading to the complete absence of
MeCP2 protein in the brain . Male mutant mice stop to grow at
4-5 weeks of age and die at 8-12 weeks (mice in the 129 background
typically die earlier than those in the mixed 129/C57BL/6 background),
while heterozygous females have the same growth profile as wildtype
littermates. These mice showed cerebellumdependent motor ability
deficits, and impaired hippocampus/amygdala-dependent behavioral
tasks, such as cued and contextual fear learning .
|To allow a more controlled deletion of Mecp2, its endogenous
transcription was disrupted by inserting a lox-STOP-Lox cassette before
exon 3 (Mecp2Stop) that can be removed by Cre-mediated recombination
[26,49]. Similar to Mecp2tm1.1B mice, these mice (C57BL/6 background)
do not show any signs of MeCP2 protein expression, and develop
identical RTT-like neurological phenotypes. Male Mecp2Stop mice
typically start to show progressive symptoms at ~6 weeks of age with
death occurring at ~11 weeks, while heterozygous females are fertile and
display neurological phenotype only after 4-12 months. A recent study
described that these mice show thinner neocortex and corpus callosum,
smaller cell body size, and shorter length of basal dendrites in layer II/
III pyramidal cells of the motor cortex than their wildtype controls .
At the synaptic level, spine density in hippocampal area CA1 is lower
compared to wildtype controls. They also show impaired long-term
potentiation (LTP) at excitatory synapses in the hippocampus, which
may lead to learning and memory deficits .
|Mecp2308 mice (either C57BL/6J or 129/SvEv backgrounds) were
generated by introducing a premature STOP codon in the Mecp2 gene,
which leads to the expression of a protein truncated at amino acid
308 containing the part of the MBD, TRD, and nuclear localization
signal . Under careful examination, male hemizygous Mecp2308
mice display more intense curling, and more pivoting and head rising
behaviors as early as postnatal day 3 . Symptoms become evident
at 6 weeks of age, which include tremors, hypoactivity, kyphosis,
myoclonic seizure, and stereotypic forelimb motion. Overall, these
symptoms are much milder than those observed in Mecp2tm1.1B and
Mecp2tm1.1J mice. Also different from those mice, male Mecp2308 mice
have normal body and brain weight, and can survive over 1 year. No
obvious histological abnormalities were found in the brain. Detailed
examination of neurons and glial cells using several markers, including
microtubule-associated protein 2 (MAP2) and glial fibrillary acidic
proteins (GFAP), did not reveal any significant differences with agematched
wildtype littermates . Consistent with those findings,
the same group reported that except for subtle changes of PSD length,
neither the dendritic arborization in layer III and V pyramidal neurons
of the frontal cortex nor the density and distribution of synaptic vesicles
are affected in male Mecp2308 mice more than 1 year old .
|Intriguingly, Mecp2308 mice exhibit upregulation of corticotrophinreleasing
hormone (CRH) and its associated heightened anxiety
[51,54,55], which differs from other Mecp2 mutant mice [44,48,56],
and hypomorph floxed mice [29,30]. The cellular bases for increased
anxiety have been related to the amygdala; consistently, both synaptic
maturation and developmental elimination are irregularly enhanced in
principal neurons of the lateral nucleus of Mecp2308 mice . Again
owing to the potentially aberrant function of truncated MeCP2 protein,
behavioral changes are often manifested in a subtle way and giving rise to some conflicting observations, such as for fear learning [51,53] and
social interaction tasks [54,58,59].
|Specific Spatio-Temporal Deletions
|Deletion in forebrain postmitotic neurons: Mecp22loxJ; CamKCre93 mice
|To generate mice with Mecp2 deletion restricted to forebrain
postmitotic neurons, Mecp2exon3-floxed (Mecp22loxJ, Jaenisch floxed)
males were crossed with females expressing Cre under control of the
CamK promoter . MeCP2 protein reduction in this conditional
mouse line starts at the perinatal phase and reaches a maximal level at
postnatal day 21. These mice have a delayed and milder phenotype than
those resulting from germline or mediated deletions . Symptoms
include hindlimb clasping, impaired diurnal activity, and cue-sensitive
Nestin-Cre-fear conditioning; however, context dependent fear
conditioning is unaffected . Weight gain, elevated anxiety, and
abnormal social interaction were also pronounced in these mice .
Histological analyses showed smaller neuronal somata in the cortex
and hippocampus, but not in the hindbrain such as cerebellum .
One of the interesting conclusions we can draw from these studies is
that the cerebellum, a brain region critical for motor control, may not
contribute to the manifestation of impaired motor coordination. Loss
of Mecp2 in broad forebrain regions and in some regions of the basal
ganglia is sufficient to cause the observed motor deficits.
|Deletion in hypothalamic neurons: Mecp22loxB; Sim1-Cre mice
|Breeding heterozygous Mecp22loxB (Bird floxed) females with males
(FVB background) expressing Cre under control of the Sim1 promoter
generated hypothalamus-specific Mecp2 conditional knockout mice
. Immunohistochemical staining demonstrated the absence of
MeCP2 protein in several primary regions of the hypothalamus,
including the paraventricular (PVN) and supraoptic nuclei (SON). As
expected from such cell-specific Mecp2 deletion, these mice recapitulate
a subset of the phenotypes observed in patients with typical or atypical
RTT. These mice show stereotypical movement, kyphosis, and increased
body weight, but lack tremor, impaired motor coordination, and
learning and memory deficits. Furthermore, hypothalamus-mediated
behaviors are impaired, resulting in enhanced anxiety, and increased
aggression in response to stress . On the basis of this study, we can
conclude that proper MeCP2 function in the hypothalamus is critical
for homeostatic responses to a variety of physiological stimuli, which
agrees with heightened anxiety and altered stress in RTT patients,
symptoms indicative of hypothalamic dysfunction.
|Deletion in dopaminergic and noradrenergic neurons
(Mecp22loxB; TH-Cre mice) and serotonergic neurons
(Mecp22loxB; PET1-Cre mice)
|To specifically remove Mecp2 from dopaminergic and noradrenergic
neurons, Mecp22loxB (Bird floxed) females were bred with males (FVB/N
background) expressing Cre under control of tyrosine hydroxylase (TH) promoter . Deletion of Mecp2 from serotonergic neurons was
achieved by Cre-mediated recombination in PC12 ets factor 1(PET1)-
expressing neurons (C57BL/6 background) . In Mecp22loxB;TH-Cre mice, MeCP2 is significantly decreased in neurons of substantia nigra or ventral tegmental area of locusceruleus. Mecp22loxB;PET1-Cre mice
show MeCP2 deficient neurons in the dorsal and medial raphe nuclei.
By comparing the phenotype between Mecp22loxB;PET1-Creknockouts,
their wildtype counterparts, and Mecp22loxB Bird floxed hypomorph
mice, Samaco et al.  found no obvious differences in several features, including breathing pattern, repetitive behaviors, anxiety levels, motor
learning and social interaction. However, conditional knockout mice
with Mecp2 deletions in dopaminergic and noradrenergic neurons
showed subtle but characteristic phenotypes, such as hypoactivity and
aggression, respectively. This study provides strong evidence for the
importance of MeCP2 in the regulation of aminergic transmitters, and
contributes to our understanding of the role of different transmitters in
|Deletion in GABAergic neurons: Mecp22loxB; Viaat-Cre and
|Deletion of Mecp2 from GABAergic interneurons was
accomplished by crossing Mecp22loxB (Bird floxed) females with males
(FVB background) expressing Cre under control of the Viaat promoter
. Mecp22loxB;Viaat-Cre mice exhibit the stereotypic neurological
phenotype observed in Mecp2tm1.1B and Mecp2tm1.1J mice, developing
symptoms at 5 weeks of age and having a half-survival of 26 weeks.
An additional number of features distinct from Bird nulls and Jaenisch
mutants include impaired sensorimotor gating and arouse (assessed with
acoustic startle response and pre-pulse inhibition tasks) and enhanced
social interests, but without altered anxiety and aggression. Interestingly,
the expression levels of the GABA synthetic enzymes glutamic acid
decarboxylase 65 (Gad65) and Gad67 are reduced in Mecp22loxB;Viaat-
Cre mice, with a concomitant reduction in vesicular GABA content
and impaired synaptic inhibition. To further dissect MeCP2 function
in forebrain interneurons, male mice (FVB background) expressing
Cre under the Dlx5/6 promoter were crossed with Mecp22loxB females
. It is worth noting that the Dlx5/6 promoter is only expressed
at high levels in parvalbumin-positive forebrain interneurons that
innervate pyramidal cell somata, but is not expressed in interneurons
that target pyramidal cell dendrites, such as calretinin positive and
somatostatin-positive interneurons . The phenotype observed in
Mecp22loxB;Dlx5/6-Cre mice thus resulted from MeCP2 deficiency only
in a subset of forebrain interneurons with a specific functional role.
Compared to the more widespread Mecp2 deletion in all interneurons
in the Mecp22loxB;Viaat-Cre mice, Mecp22loxB;Dlx5/6-Cre mice show
milder symptoms and longer life-span, surviving to over 80 weeks.
Except for decreased motility, Mecp22loxB;Dlx5/6-Cre mice show similar
phenotypic features presented by Mecp22loxB;Viaat-Cre, including
hindlimb clasping, reduced locomotor activity, kyphosis, breathing
dysfunction. . It is surprising that unlike other conditional mutants,
deletions of Mecp2 in all interneurons or even in a specific subset of
interneurons result in a multitude of RTT-like symptoms usually
observed in widespread Mecp2 deletions. A tenable conjecture will
be that perturbation of MeCP2 function even in small-scale territory
of GABAergic interneurons is enough to ruffle the neuronal network
and ultimately result in a phenotype similar to that seen in mice with a
widespread loss of MeCP2 function.
|Deletion in glial cells: Mecp22loxJ; hGFAP-CreT2 mice
|Neuronal dysfunction was initially considered the sole basis for
RTT etiology, but accumulating evidence indicates that glial cells may
also have an important role in disease pathophysiology . Contrary
to an initial belief, MeCP2 is expressed in significant levels by glial
cells, including astrocytes and oligodendrocytes . Furthermore,
astrocytes from Mecp2tm1.1J mice had detrimental consequences on
either wildtype or Mecp2-deficient neurons, an effect also observed by
astrocyte-conditioned medium . Consistently, cytokine production
altered astrocytic glutamate clearance in Mecp2tm1.1B mice [65,66].
In addition to astrocytes, microglia also shows impaired function in Mecp2tm1.1B mice, as they release excitotoxic levels of glutamate , and
do not respond properly to immunological stimuli .
|To confirm these unexpected roles of MeCP2 role in glial cells,
Mecp22loxJ (Jaenisch floxed) mice were crossed with mice (mixed
FVB/N/C57BL/6 background) harboring a tamoxifen-inducible
Cre transgene driven by the human GFAP regulatory element
(Mecp22loxJ;hGFAP-CreT2). The specific Mecp2 deletion in astrocytes
after postnatal day-21 results in a number of classical RTT-like
symptoms, including decreased body weight, hindlimb clasping, and
irregular breathing, while longevity, locomotion, and anxiety were
not affected . The observation of normal dendritic complexity in
neurons of these astrocyte-specific Mecp2 null mice appears to contrast
with earlier reports in widespread Mecp2 mutant mice; however, it
is conceivable that Mecp2 deletion in astrocytes at this later age (i.e.
P21) does not seriously compromise already established neuronal
morphology. These studies demonstrate that functional loss of glia, an
integral component critical for proper brain function, would breach the
intimate relationship with neurons and lead to the development of the
|Viral-mediated Mecp2 deletion: Cre- and shRNA-expressing
|To achieve Mecp2 deletion in a spatial as well as temporal manner,
viral-mediated approaches have been successfully employed. Targeted
Mecp2 deletion achieved by injections of Adeno-Associated Virus
(AAV) expressing Cre recombinase in the basolateral amygdala
of Mecp22loxJ mice (Jaenisch floxed) resulted in increased anxiety
and deficits in cue-dependent fear conditioning, without affecting
motor coordination, locomotor activity and social behavior .
Also, MeCP2 protein expression was knockdown with short hairpin
RNAs (shRNAs) designed to target Mecp2 mRNAs. Rat pups that
received intraventricular injections of MeCP2 shRNA show altered
sensory-motor reflexes and neurobehavioral abnormalities during
early development, but only transiently . Even though MeCP2
knockdown by shRNA injections into the dorsal striatum resulted in
decreased intake of a drug-of-abuse, RTT-like phenotypes were not
apparent . Infusions of MeCP2 shRNA-expressing lentivirus into
the Nucleus Accumbens (NAc) had a converse effect (increased drug
reward response), but also without occurrence of RTT like symptoms
. It is undoubted that new findings would emerge by using these
vial mediated knockdown techniques, which can achieve local ablation
of gene expression inaccessible to DNA promoter-driven knockout
|Tamoxifen-driven expression of Cre recombinase: Mecp22loxB;
|Through its interaction with the estrogen receptor (ER), tamoxifen
triggers the nuclear translocation of an engineered Cre-ER complex in
CAGGS-CreER mice, allowing the Cre recombinase to interact with
loxP sites flanking gene regulatory regions of interest and excise them.
Systemic tamoxifen injections allowed Mecp2 deletion at postnatal-day
60 or older Mecp22loxB; CAGGS-CreER mice (C57BL/6J background),
which resulted in characteristic RTT-like behavioral deficits and death
13 weeks after treatment onset . A follow-up study revealed that
conditional Mecp2 deletion either at 5 weeks or at 11 weeks resulted
in similar disease progression and lifespan (16-17 weeks after onset of
tamoxifen treatment) . Intriguingly, despite the RTT-like disease
progression and fatality, no differences were found at the cellular and
molecular levels, including neuronal density and soma size, dendritic
complexity, and expression of several synaptic proteins. Another study extending the age of tamoxifen onset to even earlier and later found
that, although all mice eventually reached the same disease severity by
10 weeks after tamoxifen onset, mice treated at 3 weeks-of-age develop
symptoms more stagnantly in the first week compared to mice that
received tamoxifen starting at 11- and 20-weeksof-age . It is also
interesting to note that, irrespective of ages for tamoxifen treatments,
all mice die at ~40 weeks of age. These studies suggest that MeCP2 levels
are critical during an early age (8-14 weeks) when synaptic refinement
occurs, as well as later (30-45 weeks) when aging processes initiate. Even
though this hypothesis awaits further examination, it is increasingly
clear that proper MeCP2 levels are of great importance in the adult
brain. The use of tamoxifen-induced Cre recombination has produced
fruitful results regarding the required timing of MeCP2 function. In
combination with several models of local Cre-ER expression, it has
enabled us to map the neuroanatomic origins of phenotypes observed
in RTT mouse models, shedding light on the sources of clinical
presentations in RTT individuals.
|Single Point Mutations
|Missense or nonsense MECP2 mutations, including R106W,
R133C, T158M, R168X, R255X, R270X, R294X, and R306C, account
for more than half of the RTT cases . Thus, mice carrying a single
nucleotide change in Mecp2 would represent the closest experimental
model of patients with RTT.
|Truncated MeCP2: Mecp2R168X mice
|Mecp2R168X mice (mixed C57BL/6, 129S6/SvEv Tac background)
carry a single point mutation, where the codon for arginine at position
168 is replaced with a premature STOP codon . These mice express
a truncated MeCP2 protein at ~50% wildtype MeCP2 levels. As seen
in other Mecp2 mouse lines, male mutant mice are more severely
affected than female heterozygotes. Male Mecp2 R168X mice have a
short lifespan (~12 weeks) with neurological features starting as early
as 5-6 weeks, while female heterozygotes show symptoms only after six
months of age and live longer than 1 year. Symptoms in males consist of
forelimb stereotypes, hindlimb clasping and atrophy, hypoactivity, and
breathing irregularities, while females have similar but milder signs.
Compared with two main mutant mice in phenotypic severity, they are
milder than Mecp2 tm1.1B and close to Mecp2tm1.1J mice. A note added in
proof in the Lawson-Yuen et al.  paper warned about mistakenly
introduced mutations after the STOP codon, which preclude the use
of these mice to test read-through compounds that skip STOP codons,
such as aminoglycoside antibiotics (i.e. the full-length protein will
express several mutations of unpredictable consequences).
|MECP2 mutation in males: Mecp2A140V mice
|The Mecp2A140V mouse model is unique because it mimics atypical
RTT cases occurring mostly in males. The Mecp2A140V mutation is
known to retain the methyl-CpG binding function while losing its
ability to be associated with the mental disability-related protein ATRX
. Mecp2A140 mice (mixed 129X1/S1, C57BL/6 background) show
normal levels of Mecp2 mRNA and protein, which may allow normal
lifespan, weight, and motor coordination, as well as breathing rhythms.
However, they do show some RTT-like features, such as increased
neuronal cell density, and decreased complexity of dendritic branching
in layer II/III pyramidal neurons of the somatosensory cortex .
|Phosphorylation mutants: Mecp2S421A and Mecp2S421A;S424A mice
|Post translational modifications of MeCP2 are known to have a
critical role in its binding to target genes and their ensuing expression . Phosphorylation of MeCP2 sites, especially at serine 421, has been
under intense investigation. Several studies suggested a de-repression
model in which phosphorylation of MeCP2 following neuronal activity
prevents it from binding to target genes and results in expression
of genes and the ensuing modulation of dendritic patterning and
dendritic spine morphogenesis [76-78]. This view has been revised
by a recent study using MeCP2 S421A point mutation mice (mixed
sv129, C57BL/6 background) and ChIP-Seq . These mutant mice
exhibit some cellular phenotype including an increase in the number of
dendritic branches in cortical pyramidal neurons, and a shift towards
synaptic inhibition in the excitation/inhibition. At the behavioral
level, responses to a novel mouse were significantly impaired, but
motor activity, social interaction, spatial learning and memory were
unaffected. It is surprising that, in this study using ChIP-Seq analysis
for genome wide DNA-protein association and ChIP-qPCR for specific
association, they did not find any significant changes in MeCP2 binding
to target genes. These findings suggest phosphorylation of MeCP2 at
serine 421 does not regulate specific genes, but instead plays a role in
fine-tuning global gene expression. However, the authors also pointed
out that global histone-like manipulation of MeCP2 at serine 421 does
not exclude the possibility of facilitating discrete gene expression in the
presence of other locus-specific modifications of MeCP2. This notion,
although not been tested, could be true. Interestingly, a recent study
showed that the additional substitution of serine for alanine at position
424 in Mecp2S421A;S424A mice (C57BL/6 background) resulted in enhanced
contextual fear learning, spatial memory, hippocampal synaptic
plasticity (i.e. LTP at CA3-CA1 and mossy fiber-CA3 synapses), and
synaptogenesis, while motor activity, coordination and nervousness
were unaffected . However, an earlier study using mice (C57BL/6
background) with similar mutations had shown increased locomotor
activity . The conflicting observations in these studies suggest
complex and diverse consequences of phosphorylation at different
MeCP2 sites. Phosphorylation at S421, for example, was revealed to
result in completely different outcomes as for phosphorylation at S80,
as discussed next.
|Phosphorylation mutants: Mecp2S80A mice
|Mice (C57BL/6 background) with serine 80 replaced by alanine in
MeCP2 (Mecp2S80A) to prevent phosphorylation at this site did not exhibit
any RTT-like phenotypes, except for decreased locomotor activity .
In striking contrast to phosphorylation at S421, S80 phosphorylation
was negatively regulated by neuronal activity. The finding of an
opposing regulation of S421 and S80 phosphorylation by neuronal
activity raises the intriguing possibility that MeCP2 phosphorylation is
engaged differentially under resting and active states.
|Mecp2 hypomorphs: Mecp2T158A mice
|Threonine 158 in MeCP2 is known to play a crucial role in the
stabilization of the MBD and the binding of MeCP2 to its target genes
. Since a threonine 158 mutation is common in RTT individuals,
Mecp2T158A mice (sv129, C57BL/6 background) were generated to
investigate its role in RTT pathogenesis . Male Mecp2T158A mice
develop RTT-like symptoms at 5 weeks of age and die at ~16 weeks,
a similar or slightly milder disease than that seen in Mecp2R168X mice.
Similar to all other mouse models, and likely due to the mosaicism of
an X-link mutant allele, female Mecp2T158A heterozygous mice do not
present with symptoms until 17 weeks of age, and live a normal lifespan.
In addition to the characteristic RTT-like phenotype, which includes
hypoactivity, hindlimb clasping, impaired motor coordination, and
learning & memory deficits, Mecp2T158A mice have an initial body weight loss that later subsides, a different pattern of weight variability
than that presented by Mecp2R168X male mice. Unlike the complete
absence of MeCP2 in Mecp2R168X mice and the normal levels of MeCP2
protein in Mecp2A140V, Mecp2S421A/Mecp2S421A;S424A, and Mecp2S80A mice,
symptomatic Mecp2T158A mice show ~60% lower levels of MeCP2
protein compared to age-matched littermate controls. This reduction
in MeCP2 levels is thought to underlie impaired expression of target
genes (e.g. Bdnf).
|Transgene Rescue of Mecp2 expression
|Ever since the discovery that MECP2 mutations cause RTT, despite
the recognized challenge to only target the mutant allele in a X-linked
disorder, prevention or reversal of RTT phenotypes has been attempted
by means of transgene expression in experimental mouse models. An
unexpected corollary of these studies is that elevated levels of MeCP2
are as detrimental to neurological function as their reduction .
|Neuronal Mecp2 overexpression: Tau-Mecp2; Mecp2tm1.1J
|An additional copy of the mouse Mecp2 gene was inserted into
the Tau locus (encoding the neuron-specific microtubule associated
protein Tau) to test for the rescue of RTT-like phenotypes by increasing
MeCP2 protein levels in neurons . However, it was found that
homozygous Tau-Mecp2 mice (FVB background) exhibit similar
symptoms as Mecp2tm1.1J mice (Jaenisch mutants), likely due to
excessive levels MeCP2 protein under control of the Tau promoter.
Incidentally, a similar neurological phenotype was observed in mice
that overexpressed the human MECP2 gene from a P1-derived artificial
chromosome containing all regulatory elements , demonstrating
that MeCP2 overexpression is detrimental to brain function. When
Tau-Mecp2 mice (mixed 129, C57BL/6 background) were bred with
Mecp2tm1.1J (Jaenisch mutants), their progeny showed a significant
reduction of all RTT-like phenotypes examined , suggesting that
the mild increase in MeCP2 levels from one copy of Tau locus was
sufficient to prevent disease onset but not too high to be deleterious.
|Cre-mediated Mecp2 reactivation by cell-specific promoters:
CAGGSMecp2Stop; Mecp2tm1.1J; Nestin-Cre/Tau-Cre/CamK-Cre mice
|A construct containing the synthetic CAGGS promoter, a loxP
flanked STOP cassette, and mouse Mecp2 cDNA was targeted to the
Col1a1 locus and subsequently transferred into ES cell to generate
CAGGS-Mecp2Stop mice (C57BL/6 background) . The lack of Mecp2 expression due to a STOP codon results in the characteristic RTT-like
phenotypes in CAGGS-Mecp2Stop mice. These mice were crossed with
Mecp2tm1.1Jmice (Jaenisch mutants), and the resulting offspring were
bred with different Cre-expressing transgenic mice. Removal of the
STOP codon by Cre-mediated recombination leads to reactivation of the
CAGCS-Mecp2 transgene and significant MeCP2 protein expression,
which was higher in Nestin-Cre (neural lineage) and Tau-Cre (neuronspecific
microtubule associated protein) mice than in CamK-Cre mice
(specific to forebrain neurons). Mice with higher Mecp2 transgene
expression showed longer lifespan than Mecp2tm1.1J mice without
overt RTT-like phenotypes, while mice with lower Mecp2 transgene
expression showed slight improvements compared to Mecp2tm1.1J mice.
For example, CamK-Cre rescue mice lacked the characteristic low brain
weight and small neuronal soma size observed in Mecp2 mutant mice
|CreER-mediated Mecp2 reactivation by systemic tamoxifen
treatment: Mecp2StopB; CreER mice
|As above, lack of Mecp2 expression due to a STOP codon results
in characteristic RTT like phenotypes in Mecp2StopB;CreER mice
(C57BL/6 background). Excision of the STOP cassette by tamoxifeninduced
nuclear translocation of the CreER complex results in the reexpression
of Mecp2 under control of native regulatory elements .
Compared to the rapid progression of RTT-like phenotypes in male
Mecp2StopB;CreER mice, mice receiving tamoxifen starting at 3-4 weeks
after birth showed a significant delay in symptom progression and a
longer lifespan of up to 17 weeks of age. Similar delayed onset symptoms
and extended lifespan were observed in female Mecp2StopB;CreER mice
after tamoxifen treatment.
|Tet-On-mediated human MECP2 expression in neurons:
MECP2-TREEGFP; CamK-tTA/Eno2-tTA; Mecp2308 mice
|A MECP2-TRE-EGFP construct was designed to allow for
bi-directional regulation of both human MECP2 and EGFP (for
identification of expressing cells) under the control of a central TRE (tetracycline-responsive element) . TRE activates gene transcription
once its tetO recognition sequence is bound by tTA (tetracycline
transactivator), which is given to mice orally in the drinking water.
Progenies resulting from the crossbreeding of mice containing the
MECP2-TRE-EGPF transgene with those containing the CamK-tTA or Eno2-tTA transgenes express MeCP2 in neurons of the forebrain
and the striatum and cerebellum, respectively. These inducible mice
(C57BL/6 background) were crossed to Mecp2308 mice to test for the
prevention of RTT-like symptoms. Surprisingly, cell specific expression
of Mecp2 failed to prevent most of the phenotypes characteristic of
Mecp2308 mice, except for a marginal increase in motor activity in
CamK rescue mice (forebrain neurons). However, earlier studies
described that specific manipulations of Mecp2 expression in forebrain
neurons resulted in detectable, albeit mild, behavioural consequences
[43,60,86]. The expression of a partially functional truncated protein
in Mecp2308 mice could contribute to this discrepancy. Also, normal
levels of Mecp2 transgene expression may increase MeCP2 protein
dosage too much, and thus compromise any potential beneficial effects.
Indeed, using a similar strategy, female heterozygous Mecp2tm1.1B mice
with optimal Mecp2 transgene expression showed improved rearing
and locomotor activity .
|CreER-mediated Mecp2 reactivation in glial cells by systemic
tamoxifen treatment: Mecp2Stop; hGFAP-Cre mice
|Glial-specific reactivation of endogenous Mecp2 silenced by a loxPflanked
STOP codon was achieved by excising it with a tamoxifeninducible
CreER placed under control of the human hGFAP promoter
. Despite lack of Mecp2 expression in neurons, Mecp2 reactivation
exclusively in glial cells extended the lifespan to 7.5 months compared
with only 3 months in oil-treated Mecp2Stop;hGFAP-Cre mice (C57BL/6
background). Tamoxifen-treated Mecp2Stop;hGFAP-Cre mice show
increased activity levels, decreased anxiety, while the characteristic
irregular breathing (apnea) frequency is also improved compared
to oil-injected controls. Consistent with these improvements in
behavioural features, the size of neuronal somata dendritic arborization,
and VGLUT1 protein expression were all significantly increased in
tamoxifen-treated Mecp2Stop;hGFAP-Cre mice.
|Introduction of wildtype microglia into Mecp2 null brain:
bone marrow transplants in Mecp2Stop; Lysm-Cre mice
|Since Mecp2-deficient microglia has adverse effect on wildtype neurons in co-cultures , it is reasonable to speculate that
repopulation of Mecp2-containing microglia would improve RTTlike
symptoms in Mecp2 deficient hosts. Indeed, a recent study
demonstrated that transplantation of wildtype bone marrow into
presymptomatic Mecp2 null mice (postnatal day 28) after whole-body
irradiation-mediated immune ablation resulted in engraftment of
microglia-like myeloid cells into the brain parenchyma and arrested the
progression of neuropathology including recovery of body and brain
size, enhancement of lifespan, improvement of gait and locomotor
activity (but not hindlimb clasping), and reduction of apneas and
breathing irregularities . However, no significant rescue was
observed when bone marrow transplants were performed during the
time RTT-like phenotypes begin to appear (postnatal day 40 or 45).
Phenotypic prevention also failed if irradiation did not include the
brain, which leads to reconstitution of only peripheral but not brain
microglia-like myeloid cells. To further support this strategy, Lysm-
Cre mice (mixed 129, C57BL/6 background), which express Cre under
control of the Lysm promoter, were crossed with Mecp2Stop mice to
generate progeny with normal Mecp2 expression by removal of STOP
codon only in myeloid cells, granulocytes, and microglia. Such cellspecific
Mecp2 reactivation was sufficient to improve breathing patterns,
prolong lifespan, and increase both body weight and locomotor activity.
Furthermore, long-term treatment of Mecp2Stop;Lysm-Cre mice with
annexin-V, a pharmacological manipulation that blocks phagocytic
activity of microglia, failed to prevent RTT-like disease progression,
strongly suggesting that micrglia dysfunction contributes to RTT
|The various experimental mouse models described above have
enabled researchers to discover novel mechanisms at the molecular,
cellular, and circuit levels that contribute to MeCP2 dysfunction in
RTT, providing remarkable platforms for testing a wide variety of
genetic, pharmacological, and physiological interventions to improve
the quality of life in this debilitating disease. Notwithstanding, and
considering the significant differences between research rodents
and humans at all levels, there is great need to establish consistent
and effective human-based models. Undoubtedly, the use of patientspecific
human induced pluripotent stem stem cells (iPSCs) is by far the
closest model experimental research and manipulations . Neuronal
differentiation of human iPSCs derived from RTT individuals has
been successfully achieved [89-94], which one day should allow highthroughput
screening of pharmacological libraries. With RTT research
evolving into full-fledged endeavors in an ever-growing number of
research laboratories, we hope that the scientific knowledge gained
from these and new animal models will be translated into rational
therapies available for RTT individuals in not too distant future.
|This work was supported by NIH grants NS-065027 and NS-40593 (LPM),
and by Postdoctoral Fellowship IRSF-2824 (WL) from the International Rett
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